Human interleukin 13 (hIL13) is a 114 amino acid cytokine secreted by activated T cells. Minty et al. (1993) Nature, 362:248-250; and McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA, 90:3735-3739. hIL13 is involved in regulating several different physiological responses. Among these, hIL13 has been shown to downregulate the production of cytokines involved in inflammation. Minty et al., supra; and de Waal Malefyt et al. (1993) J. Immunol., 151:6370-6381. It has also been shown to upregulate expression of major histocompatibility class II molecules and CD23 on monocytes, and to regulate various aspects of B cell function De Waal Malefyt et al. (1993) Res. Immunol. 144:629-633; McKenzie et al., supra; and de Waal Malefyt et al. (1993) J. Immunol., 151:6370-6381. In addition to regulating cells of the immune system, IL-13 has also been shown to act on other cell types. For example, IL13 has been shown to modulate expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Sironi et al. (1994) Blood, 84:1913-1921; Bochner et al. (1995) J. Immunol., 154:799-803; and Schnyder et al. (1996) Blood, 87:4286-4295.
Based on its predicted secondary structure, hIL13 has been added to a growing family of growth hormone-like cytokines that all exhibit bundled alpha-helical core topology. Bamborough et al. (1994) Prot. Engin,. 7:1077-1082. Structural analyses indicated that hIL13 is a globular protein comprised mainly of four alpha-helical regions (helices A, B, C, and D) arranged in a xe2x80x9cbundled core.xe2x80x9d Miyajima et al. (1992) Ann. Rev. Immunol., 10, 295-331.
While dissimilar at the primary amino acid level, hIL13 and human interleukin 4 (hIL4) bind and signal through a shared receptor complex. Zurawski et al. (1993) EMBO J., 12:2663-2670; and Tony et al. (1994) Eur. J. Biochem., 225:659-66. This shared receptor is a heterodimer that includes a first subunit of approximately 140 kDa termed p 140, and a second subunit of approximately 52 kDa termed axe2x80x2 or IL13Ra1. Idzerda et al. (1990) J. Exp. Med., 173:861-873; Obiri et al. (1995) J. Biol. Chem., 270:8797-8804; Hilton et al. (1996) Proc. Natl. Acad. Sci. USA, 93:497-501; and Miloux et al. (1997) FEBS Letters, 401:163-166. Unlike hIL4, hIL13 does not bind p140 in the absence of axe2x80x2. Vita et al. (1995) J. Biol. Chem., 270:3512-3517. In addition to the shared receptor, another hIL13 receptor termed the restricted (IL4 independent) receptor exists. In contrast to the shared receptor, the latter receptor binds hIL13 but not hIL4. The restricted receptor is also sometimes called the glioma-associated receptor because it is preferentially expressed at high levels in certain malignant cells, including those in high grade human gliomas. Debinski et al. (1995) Clin. Cancer Res., 1:1253-1258; and Debinski et al. (1996) J. Biol. Chem., 271, 22428-22433. In addition to being associated with malignancies, hIL13 has also been associated with other pathological conditions. Notably, IL13 has been shown to be involved in pathways that regulate airway inflammation, suggesting that this cytokine might play an important role in asthma and perhaps other allergic pathologies. Webb et al., (2000) J. Immunol.165:108-113; and Djukanovic, R. (2000) Clin. Exp. Allergy 30 Suppl 1:46-50.
The invention relates to the development and characterization of several mutants of hIL13. Using these mutants, three regions of native hIL13 were identified as being required for signaling through the shared receptor. These regions were localized to alpha-helices A, C and D and were generally separated from the regions involved in binding to the restricted receptor. Glutamic acids at positions 13 and 16 in hIL13 alpha-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signaling because these mutations resulted in the loss and/or gain of functional phenomena.
Mutants within the invention include those having one or more of the native amino acids of hIL13 at positions 13, 16, 17, 66, 19, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114 replaced with a different amino acid. These mutants are expressed herein as hIL13.X1PX2, where P is a number corresponding to the position of the mutated amino acid in hIL13, X1 is the letter abbreviation of the amino acid that was replaced, and X2 is the letter abbreviation of the replacement amino acid. Mutants with multiple mutations are indicated in the same fashion as hIL13X1PX2.X3P1X4 for a double amino acid substitution mutant hIL13X1PX2.X3P1X4.X5P2X6 for a triple amino acid substitution mutant; and hIL13X1PX2.X3P1X4.X5P2X6.X7P3X8 for a quadruple amino acid substitution mutant. For example, hIL13.E13K represents a mutant form of hIL13 that has the glutamic acid residue that naturally occurs at position 13 in native hIL13 replaced with a lysine residue; and hIL13.E13K.S69D represents a mutant form of hIL13 that has the glutamic acid residue that naturally occurs at position 13 in native hIL13 replaced with a lysine residue and the serine residue that naturally occurs at position 69 in native hIL13 replaced with an aspartic acid residue. Representative single amino acid substitution mutants within the invention include hIL13.E13K, hIL13.E13I, hIL13.E13C, hIL13.E13S, hIL13.E13R, hIL13.E13Y, hIL13.E13D, hIL13.E16K, hIL13.E17K, hIL13.R66D, hIL13.S69D, hIL13.D99K, hIL13.L102A, hIL13.L104A, hIL13.K105D, hIL13.K106D, hIL13.L107A, hIL13.F108Y, hIL13.R109D, hIL13.R112D, hIL13.F113D, and hIL13.N114D. The invention also includes double, triple, and quadruple amino acid substitution mutants including: hIL13.E13K.S69D (SEQ ID NO:2); hIL13.E13K.R109D (SEQ ID NO:3); hIL13.E13K.R112D (SEQ ID NO:4); hIL13.E13Y.R66D (SEQ ID NO:5); hIL13.E13Y.S69D (SEQ ID NO:6); hIL13.E13K.R66D.S69D (SEQ ID NO:7); hIL13.E13Y.R66D.S69D (SEQ ID NO:8); and hIL13.E13K.R66D.S69D.R112D (SEQ ID NO:9).
Accordingly, the invention features a purified mutant hIL13 molecule including an amino acid sequence (a) having at least 90% sequence identity to the native hIL13 sequence (SEQ ID NO:1) and (b) differing from the native hIL13 sequence by at least a first amino acid substitution occurring in the A alpha helix and a second amino acid substitution occurring in the D alpha helix.
Also within the invention is a purified mutant hIL13 molecule including an amino acid sequence (a) having at least 90% sequence identity to the native hIL13 sequence (SEQ ID NO:1) and (b) differing from the native hIL13 sequence by at least three amino acid substitutions. In one variation of the foregoing, the amino acid sequence differs from the native hIL13 sequence by at least a first amino acid substitution occurring in the A alpha helix, a second amino acid substitution occurring in the D alpha helix, and a third amino acid substitution occurring in the C alpha helix. In another variation of the foregoing, the amino acid sequence differs from the native hIL13 sequence by at least four amino acid substitutions, e.g., with at least a first amino acid substitution occurring in the A alpha helix, a second amino acid substitution occurring in the D alpha helix, and a third amino acid substitution occurring in the C alpha helix.
The invention further includes a purified mutant hIL13 molecule that includes a polypeptide having or consisting of an amino acid sequence of one of SEQ ID NOs: 2-9.
The purified mutant hIL13 molecule of the invention can further include a pharmaceutically acceptable carrier and/or can be conjugated to an effector molecule such as a cytotoxin (e.g., a Pseudomonas exotoxin such as PE38QQR, PE1E, and PE4E, Diptheria toxin, ricin, abrin, saporin, and pokeweed viral protein), a detectable label, an antibody, a liposome, and a lipid. The effector molecule can also be a radionuclide.
In another aspect, the invention features a purified nucleic acid encoding a polypeptide including or consisting of an amino acid sequence of one of SEQ ID NOs: 2-9.
The invention additionally features an antibody that specifically binds an hIL13 mutant but not a native hIL13. The hIL13 mutant can be one of the above-described mutant hIL13 molecules such as one that includes an amino acid sequence of one of SEQ ID NOs: 2-9.
In still another aspect, the invention includes a method of delivering a hIL13 mutant to a cell. This method includes the steps of: (a) providing a hIL13 mutant (such as one described above) (b) providing the cell; and (c) contacting the cell with the hIL13 mutant. In the method, the hIL13 mutant can be conjugated to an effector molecule. The step (c) of contacting the cell with the hIL13 mutant can takes place in an animal, e.g., by administering the mutant to the animal by injection or other means. 51. Also in another variation of the method, the cell can form part of a tumor in an animal such that the tumor is growing at a measurable rate in the animal. In this variation, the rate is decreased subsequent to the step (c) of contacting the cell with the hIL13 mutant.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of molecular biology terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
As used herein, the phrase xe2x80x9cnative hIL13xe2x80x9d means the mature form of human interleukin 13, the amino acid sequence of which is shown herein as SEQ ID NO:1.
The phrase xe2x80x9chIL13 mutant,xe2x80x9d xe2x80x9cmutant hIL13,xe2x80x9d or a xe2x80x9cmutant hIL13 moleculexe2x80x9d means an hIL13 in which one or more of the amino acids differ from the corresponding amino acids in the native hIL13. Thus, for example, where a native hIL13 has a glutamic acid at position 13, a mutant hIL13 can have an amino acid other than glutamic acid at position 13 (e.g., glutamic acid is substituted with lysine). It will appreciated that mutant IL13 molecules of this invention include mutant IL13 molecules of other mammalian species (e.g., rat, murine, porcine, ovine, goats, non-human primates, bovine, canus, and the like) and this invention contemplates the use of mutant IL13 in veterinary as well as human medical conditions.
As used herein, the terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. An xe2x80x9cpurifiedxe2x80x9d polypeptide is one that has been substantially separated or isolated away from other polypeptides in a cell, organism, or mixture in which the polypeptide occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
As used herein, a xe2x80x9cnucleic acidxe2x80x9d or a xe2x80x9cnucleic acid moleculexe2x80x9d means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). A xe2x80x9cpurifiedxe2x80x9d nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 1000% free of contaminants). The term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A xe2x80x9crecombinantxe2x80x9d nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
As used herein, xe2x80x9csequence identityxe2x80x9d means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an alanine in each of two polypeptide molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 amino acids in length are identical to the corresponding positions in a second 10 amino acid sequence, then the two sequences have. 70% sequence identity. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
By the term xe2x80x9cantibodyxe2x80x9d is meant an immunoglobulin as well as any portion or fragment of an immunoglobulin whether made by enzymatic digestion of intact immunoglobulin or by techniques in molecular biology. The term also refers to a mixture containing an immunoglobulin (or portion or fragment thereof) such as an antiserum.
The term xe2x80x9cspecifically bindsxe2x80x9d, as used herein, when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody), the specified ligand or antibody binds to its particular xe2x80x9ctargetxe2x80x9d (e.g. an IL13 specifically binds to an IL13 receptor) and does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism. Generally, a first molecule that xe2x80x9cspecifically bindsxe2x80x9d a second molecule has a binding affinity greater than about 105 (e.g., 106, 107, 108, 109, 1010, 1011, and 1012 or more) moles/liter for that second molecule.
A xe2x80x9cmutationxe2x80x9d in a polypeptide refers to the substitution of an amino acid at a particular position in a polypeptide with a different amino acid at that position. Thus, for example, the mutation hIL13.E13K.S69D indicates that the native amino acids at positions 13 and 69 in IL13 (glutamic acid, E; and serine, S) are replaced with lysine (K) and aspartic acid (D) respectively. In some cases, a mutation can be the deletion, addition, or substitution of more than one amino acid in a polypeptide. The mutation does not require an actual removal and substitution of the amino acid(s) in question. The protein can be created de novo with the replacement amino acid in the position(s) of the desired mutation(s) so the net result is equivalent to the replacement of the amino acid in question.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials, are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.